Interpretive Handbook

The plasma membranes of mammalian cells are formed from phosphorlipids. Anionic phospholipids (eg, phosphatidylserine) are found on the cytoplasmic surface and neutral phospholipids (eg, phosphatidylcholine) predominate on the external surface. Membrane phospholipids participate in several important cellular functions including exchanging metabolites across membranes, transferring molecular signals and serving as a platform for the assembly of protein-lipid complexes.(1) Cellular activation is often accompanied by the translocation of anionic phospholipids to the external membrane surface. For example, during platelet-mediated blood coagulation phosphatidylserine is translocated from the inner platelet membrane and provides a surface for the assembly of the prothrombinase enzyme complex that catalyzes the formation of thrombin.

Complexes of negatively charged (anionic) phospholipids and endogenous plasma proteins provide epitopes recognized by natural autoantibodies.(2) Plasma from normal individuals contains low concentrations of natural IgG autoantibodies of moderate affinity. Pathologic levels of autoantibodies reflect loss of tolerance and increased production of antibodies. These autoantibodies are called phospholipid or cardiolipin antibodies when they are detected by immunoassays that employ anionic phospholipids as substrates. The most commonly used phospholipid substrate is cardiolipin. The term phospholipid antibody is actually a misnomer. The autoantibodies react with epitopes of protein molecules that associate noncovalently with reagent phospholipids. The best characterized phospholipid-binding protein is beta 2 glycoprotein 1 and most immunoassays for phospholipid antibodies employ a composite substrate consisting of cardiolipin plus beta 2 glycoprotein 1(beta 2 GP1). Beta 2 GP1 is a 326 amino acid polypeptide that contains 5 homologous domains of approximately 60 amino acids each. Most phospholipid antibodies bind to an epitope associated with domain 1 near the N-terminus. Autoantibodies can also be detected by the use of functional, phospholipid-dependent coagulation assays. Phospholipid antibodies detected by functional assays are often called lupus anticoagulants because they produce prolongation of phospholipid-dependent clotting in vitro. Not all phospholipid antibodies possess lupus anticoagulant activity.(3) Only those phospholipid antibodies that are capable of cross-linking beta 2 GP1 molecules can interact efficiently with phospholipid surfaces in functional coagulation assays. It is hypothesized that complexes formed in vivo between bivalent, natural autoantibodies and beta 2 GP1 bind to translocated, anionic phospholipid on activated platelets at sites of endothelial injury. This binding is believed to promote further platelet activation that may lead to thrombosis.

Phospholipid antibodies occur in patients with a variety of clinical signs and symptoms notably thrombosis (arterial or venous) pregnancy morbidity (unexplained fetal death, premature birth, severe preeclampsia, or placental insufficiency) unexplained cutaneous circulation disturbances (livido reticularis or pyoderma gangrenosum) thrombocytopenia or hemolytic anemia and nonbacterial thrombotic endocarditis. Phospholipid antibodies and lupus anticoagulants are found with increased frequency in patients with systemic rheumatic diseases especially lupus erythematosus. The term antiphospholipid syndrome (APS) or Hughes' syndrome is used to describe the triad of thrombosis, recurrent fetal loss and thrombocytopenia accompanied by phospholipid antibodies or a lupus anticoagulant. The diagnosis of APS requires 1 or more of the above mentioned clinical findings plus positive test results for phospholipid antibodies (> or =40 GPL or MPL) or positive tests for a lupus anticoagulant on more than 1 occasion separated by at least 6 weeks.(4)

Testing for phospholipid antibodies is indicated in the following clinical situations:

-Unexplained arterial or venous thrombosis

-A history of pregnancy morbidity defined as 1 or more unexplained deaths of a morphologically normal fetus beyond the 10th week of gestation, 1 or more premature births before 34 weeks of gestation caused by severe preeclampsia or placental insufficiency, or 3 or more unexplained, consecutive spontaneous abortions before the 10th week of gestation with no identifiable maternal hormonal or anatomic, or maternal or paternal chromosomal causes

Positive and strongly positive results for phospholipid antibodies (> or =40 GPL and/or MPL) are a diagnostic criterion for antiphospholipid syndrome (APS). Lesser levels of phospholipid antibodies and antibodies of the IgA isotype may occur in patients with clinical signs of APS but the results are not considered diagnostic.

Detection of phospholipid antibodies is not affected by anticoagulant treatment.

The immunoassay for phospholipid antibodies does not distinguish between autoantibodies and antibodies produced in response to infectious agents or as epiphenomena following thrombosis. For this reason, a single positive test result is not sufficient to meet accepted serologic criteria for the diagnosis of antiphospholipid syndrome (APS).

Comparative studies and interlaboratory proficiency surveys indicate that results of phospholipid antibody tests can be highly variable and results obtained with different commercial immunoassays may yield substantially different results.(5,6)